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論文中文名稱:鏈黴菌線型質體SCP1接合傳遞的cis-acting element [以論文名稱查詢館藏系統]
論文英文名稱:Identification of a cis-acting element on Streptomyces linear plasmid SCP1 for conjugal transfer [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
出版年度:97
中文姓名:許嘉津
英文姓名:Chia-Chin Hsu
研究生學號:95688002
學位類別:碩士
語文別:中文
口試日期:2008-07-30
論文頁數:63
指導教授中文名:黃志宏
口試委員中文名:陳文盛;楊千金
中文關鍵詞:鏈黴菌接合生殖SCP1接合生殖傳遞的起始點
英文關鍵詞:StreptomycesconjugationSCP1oriT
論文中文摘要:鏈黴菌具有線型的染色體與質體,在其末端上會有末端蛋白質以共價鍵鍵結於DNA的5’端。鏈黴菌的部分線型質體,具有將自己驅動從供給株傳遞到接受株的接合生殖能力,並且也能驅動線型染色體傳遞的接合生殖。現今已知細菌接合生殖的機制大部分都來自大腸桿菌染色體及其F質體的研究結果,這兩者的DNA構型都是環型的,但並不適用於鏈黴菌線型的DNA的接合生殖機轉。對於這些線型染色體與質體的接合傳遞的分子機制了解甚少。因此,若能找出鏈黴菌線型質體接合生殖傳遞的起始點,將有助於解開線型DNA接合生殖機制的謎。我使用非常有效率的同源重組系統,將SCP1與構築好的同源線形質體發生交換後,在鏈黴菌活體內,產生兩個各帶有SCP1一臂與線形質體一臂不同長度的重組線型質體為策略,然後去測試在SCP1驅動下,這兩個重組的線型質體的接合傳遞能力,看看其中哪段DNA包含SCP1的oriT,而具有被接合傳遞的功能。在使用這些由SCP1發生不同位置的交換的重組質體為策略下,我已把oriT可能所在的區域縮短至SCP1上的一個約2.4 kb大小的DNA片段,由這個2.4 kb的DNA片段所構築的質體可以在SCP1的驅動下,被接合傳遞到接受株中。
論文英文摘要:The linear replicons of Streptomyces carry covalently-bound terminal proteins (TPs) at their 5’ end of DNA. Some of the linear plasmids of Streptomyces possess the ability of conjugal transfer from a donor to a recipient. These plasmids also drive the transfer of the linear chromosomes of Streptomyces. The current established mechanism of conjugation is mostly based on circular DNA in E. coli, which cannot be applied to linear replocons. The molecular mechanism of conjugal transfer of the linear replicons of Streptomyces is unknown. Identification of the origin of conjugal transfer of Streptomyces linear plasmids would help understand the conjugation mechanism. I used efficient homologous recombination to a construct truncated variants of SCP1 plasmids in vivo. Then we tested the transfer ability of these truncated plasmids driven by SCP1. A truncated SCP1 variant plasmid could be transferred to recipient, if it contained the oriT of SCP1. By this strategy, we narrowed the oriT of SCP1 down it to an internal 2.4-kb sequence.
論文目次:目 錄

中文摘要..................................................................................................................... i
英文摘要.................................................................................................................... ii
誌謝........................................................................................................................... iii
目錄........................................................................................................................... iv
表目錄........................................................................................................................ v
圖目錄....................................................................................................................... vi
第一章 緒論.............................................................................................................. 1
第二章 材料與方法................................................................................................ 14
第三章 實驗結果.................................................................................................... 19
第四章 討論…........................................................................................................ 44
參考文獻.................................................................................................................. 50
附錄
Appendix A Media and buffer ................................................................................. 54
Appendix B E.Coli Competent cell preparation and transformation........................ 55
Appendix C Plasmid isolation from E.coli............................................................... 58
Appendix D Preparation of Streptomyces protoplast and transformation with plasmid DNA............................................................................................................ 59
Appendix E Isolation Streptomyces total DNA........................................................ 60
Appendix F PCR (Polymerase Chain Reaction)....................................................... 61
Appendix G Pulsed-Field Gradient gel Electrophoresis........................................... 62

表目錄

表1.1 大腸桿菌F質體轉移區的一系列tra基因(transfer gene)的功能..............3
表2.2 菌種及質體陳列表......................................... ….14
表3.3.1 Transfer properties of linear plasmid SCP1 derivatives in S. lividans........43

圖目錄

圖1.1 大腸桿菌的F質體................................................................................. 3
圖1.2 大腸桿菌Hfr染色體與F質體的接合生殖的模型.................................. 4
圖1.3 鏈黴菌S. coelicolorA3(2)生長史.............................................................6
圖1.4 鏈黴菌的線狀染色體及線狀質體的結構................................................8
圖1.5 鏈黴菌的巨型線狀質體SCP1的結構.....................................................9
圖1.6 構築微型線狀質體pQC48的方法.........................................................11
圖1.7 兩線型質體同源片段之間的重組現象..................................................13
圖3.1.1 微型質體pLUS891、pLUS892與pLUS892rL的Hind III、BamH I限制酶圖譜與構築........................................................................................... 21
圖3.1.2 pLUS892L與SCP1同源重組的預期圖譜與結果..............................22
圖3.1.3 pLUS892r與SCP1同源重組的預期圖譜.......................................... 23
圖3.1.4 pLUS892r與SCP1同源重組的結果及其與S. lividans TK54接合生殖所得的接合轉型株分析的結果......................................................................... 24
圖3.1.5 第二次重複所得的pLUS892r與SCP1同源重組的結果及其與S. lividans TK54接合生殖所得的接合轉型株分析的結果................................... 25圖3.2.1 繼續將128 kb的SCP1’-L 縮小範圍(narrow down)找conjugal cis-element策略圖........................................................................................... 30圖3.2.2 以限制酶檢測SCP1’1(2.4 kb)與SCP1’2(2.45 kb)...............................31
圖3.2.3 pBluII KS(-)-SCP1’1與pBluII KS(-)-SCP1’2的構築..........................32
圖3.2.4 pLUS891-SCP1'-1r與pLUS891-SCP1'-1的構築.................................33
圖3.2.5 pLUS891-SCP1'-2r與pLUS891-SCP1'-2的構築.................................34
圖3.2.6 線型化質體pLUS891-SCP1'-1rL、pLUS891-SCP1'-1L、pLUS891-SCP1'-2rL與pLUS891-SCP1'-2L...................................................... 35
圖3.2.7 pLUS891-SCP1'-1rL與SCP1同源重組與作為給予株去與沒有SCP1的接受株接合生殖的接合轉型株圖譜及PFGE圖............................................ 36
圖3.2.8 pLUS891-SCP1'-1L與SCP1同源重組與作為給予株去與沒有SCP1的接受株接合生殖的接合轉型株圖譜及PFGE圖.............................................37
圖3.2.9 pLUS891-SCP1'-2rL與SCP1同源重組與作為給予株去與沒有SCP1的接受株接合生殖的接合轉型株圖譜及PFGE圖.............................................38
圖3.2.10 pLUS891-SCP1'-2L與SCP1同源重組與作為給予株去與沒有SCP1的接受株接合生殖的接合轉型株圖譜及PFGE圖.............................................39
圖3.3.1 將SCP1'-1rL的重組株cT3-1rL作為給予株去與沒有SCP1的接受株S. coelicolor J1501接合生殖的策略圖與篩選的接合生殖株cJT-1rL的PFGE圖
........................................................................... ….41
圖3.3.2 pLUS891-SCP1'-1rL的接合生殖株cJT-1rL AseI限制酶切割PFGE圖
.......................................................................................................................... 42
圖4.1 鏈黴菌線型DNA在接合生殖過程中DNA轉移的“End first model”.....49
圖4.2 假設的SCP1接合生殖的模式………………………………………....49
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論文全文使用權限:同意授權於2009-02-01起公開